PROJECT SUMMARY/ABSTRACT RNA binding proteins (RBPs) are major controllers of gene expression through post- transcriptional gene regulation (PTGR) and have been implicated in the onset of numerous disorders, including neurodegenerative diseases and cancer progression; however, the coordinated control of PTGR on a genome-wide scale remains poorly understood. According to recent surveys of the RNA interactome, the human genome is estimated to encode ~1500 RBPs, of which ~600 specifically bind mRNAs. Application of newly developed next-generation sequencing tools, such as PAR-CLIP, that enable the mapping of RBPs on a genome-wide scale, revealed that a single RBP can bind up to 50% of expressed mRNAs. Given the multiplicity of RBPs and their propensity to target significant fractions of the transcriptome, a single transcript is often bound by multiple RBPs. As such, the final outcome for PTGR is dependent on the combinatorial control of the RBPs bound to a transcript. Interestingly, RBPs targeting the same transcript do not always act independently from each other. Several instances of RBPs either cooperatively or competitively binding mRNAs to control PTGR have been reported in the literature. However, these studies identified coordinated PTGR by only a few RBPs and is therefore a highly focused observation of their activity. It remains unknown how RBPs coordinate combinatorial control of PTGR on a genome-wide scale. Considering their clinical relevance to numerous diseases, there is a critical need to identify regulatory interactions of RBPs on a genomic scale. Without such information, scientific understanding of the link between the disease states and mechanisms of PTGR will be incomplete. In Aim 1 every publicly available and in-house generated PAR-CLIP dataset will be used to identify RBPs that co-target similar sets of mRNAs or bind co-localized sites along mRNAs. Data analysis will be carried out using an established PAR-CLIP analysis pipeline in combination with custom analytical scripts. In Aim 2 the mechanism of coordinated combinatorial PTGR will be determined for candidate pairs of RBPs through RNA-seq, co-immunoprecipitation and in situ co-localization experiments. The first pair of RBPs to be characterized, METTL1 and CDK1, were already identified through our preliminary analysis of publicly available PAR-CLIP data. These results will provide a framework for the analysis of PTGR by the coordinated action of RBPs, thereby providing critical knowledge to researchers studying the mechanism or regulatory system of clinically important genes.